Factor VII, which is involved in the clotting cascade has proven to be a useful therapeutic agent to treat a variety of pathological conditions. Accordingly, there is an increasing need for formulations comprising activated Factor VII polypeptides that are pharmaceutically acceptable and exhibit a uniform and predetermined clinical efficacy. For certain therapeutic applications, it is necessary to administer a fairly large amount of an activated Factor VII polypeptide, e.g. in the order of 40 μg/kg body weight or even more.
The current commercially available, recombinantly-made Factor VII polypeptide composition NovoSeven® (Novo Nordisk A/S, Denmark), is, e.g., presented as a vial (about 3.0 mL container volume) containing 1.2 mg recombinant human Factor VIIa, 5.84 mg NaCl, 2.94 mg CaCl2, 2H2O, 2.64 mg GlyGly, 0.14 mg polysorbate 80, and 60.0 mg mannitol. This product is reconstituted to pH 5.5 by 2.0 mL water for injection (WFI) prior to use, thus yielding a concentration of the Factor VII polypeptide of about 0.6 mg/mL.
For therapeutic applications where administration of larger amounts (e.g. 10-20 mg) of an activated Factor VII polypeptide (e.g. recombinantly-made human Factor VIIa) is necessary, it is inconvenient to utilize a formulation like the NovoSeven® composition, because a fairly large volume (e.g. 15-30 mL) needs to be administered, typically by injection.
Thus, there is a need for liquid pharmaceutical products, as well as freeze-dried pharmaceutical products for subsequent reconstitution, comprising an activated Factor VII polypeptide in which a relatively high amount of the activated Factor VII polypeptide is present within a certain container volume, whereby the volume to be administered, typically by injection, causes a minimum of inconvenience for the end-user.
WO 03/055512 A1 discloses a liquid, aqueous composition comprising (i) a factor VII polypeptide; (ii) an agent suitable for keeping pH in the range of from about 4.0 to about 8.0; (iii) an agent selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; wherein the concentration of (iii) is at least 15 mM. The invention disclosed in the international patent application appears to be applicable for Factor VII polypeptides in various concentration ranges, including a concentration range of from about 0.1 mg/mL to about 10 mg/mL.
WO 2004/000347 A1 i.a. discloses a composition which, when dissolved in water, comprises a Factor VII polypeptide (0.6 to 10 mg/mL), calcium chloride (5 to 20 mM), NaCl (0-50 mM), glycylclycine (0-15 mM), L-histidine (0-20 mM), mannitol (20-40 mg/mL), sucrose (5-20 mg/mL), methionine (0-1 mg/mL), poloxamer 188 (0.5-3 mg/mL), at a pH in the range of 5.0 to 7.0.
WO 2004/082708 A1 discloses a liquid, aqueous pharmaceutical composition comprising a Factor VII polypeptide (0.1-10 mg/mL) and a buffering agent suitable for keeping pH in the range of from about 5.0 to about 9.0; wherein the molar ratio of non-complexed calcium ions (Ca2+) to the Factor VII polypetide is lower than 0.5.
WO 2004/112828 A1 discloses a liquid, aqueous composition comprising (i) a factor VII polypeptide; (ii) an agent suitable for keeping pH in the range of from about 4.0 to about 8.0; (iii) an agent selected from the list of: a calcium salt, a magnesium salt, or a mixture thereof; wherein the concentration of (iii) is less than 15 mM; and (iv) an ionic strength modifying agent; wherein the ionic strength of the composition is at least 200 mM. The invention disclosed in the international patent application appears to be applicable for Factor VII polypeptides in various concentration ranges, including a concentration range of from about 0.1 mg/mL to about 10 mg/mL.
WO 2005/016365 A1 discloses a liquid, aqueous pharmaceutical composition comprising at least 0.01 mg/mL of a Factor VII polypeptide (i); a buffering agent (ii) suitable for keeping pH in the range of from about 5.0 to about 9.0; and at least one stabilising agent (iii) comprising a —C(═N—Z1—R1)—NH—Z2—R2 motif (e.g. a benzamidine or an arginine). The invention disclosed in the international patent application appears to be applicable for Factor VII polypeptides in various concentration ranges, including a concentration range of 0.01-20 mg/mL.
Although several of the applicant's prior patent applications encompass the option of utilizing the respective invention for liquid solutions of a Factor VII polypeptide in fairly broad concentration range, the working examples thereof have not explored the upper region of such concentration ranges, neither has any of the prior patent applications disclosed or anticipated a closed container comprising a relatively high amount of an activated Factor VII polypeptide. It is believed that this is due to the general understanding that the degree of degradation of a Factor VII polypeptide, in particular heavy-chain degradation, will increase dramatically with increasing Factor VII polypeptide concentration, regardless of the general measures applied with the purpose of reducing the autocatalytic degradation of the activated Factor VII polypeptide, and that a high degree of degradation will yield products that are unacceptable for most therapeutic applications.
Furthermore, it is believed that the fact that the prior art commercial product comprises relatively high amounts of calcium may become problematic when large amounts of the active ingredient (the Factor VII polypeptide) are necessary for the treatment of particular conditions. High amounts of calcium may therefore necessitate high concentrations of Factor VII polypeptide in a small volume.
To be able to obtain a closed container with a high content of an activated Factor VII polypeptide per volume unit of the container, a highly concentrated bulk of purified, activated Factor VII polypeptide that can be dispensed into the container is required. Up till now this has been considered difficult or even impossible because activated Factor VII polypeptide is a serine protease that possesses autoproteolytic activity, which means that the protein catalyses the degradation of itself (see e.g. “The Use of RP-HPLC for measuring activation and cleavage of rFVIIa during purification” by I. Mollerup et al., Biotechnol. Bioeng., vol. 48 (1995), 501-505 and “Studies on coagulation factor VIIa autoproteolysis and formation of degradation products' by T. B. Hansen et al., poster presented at ACS (2003)”. The degradation is caused by cleavage in the C-terminal side of Lys38, Arg290 and Arg315. By cleavage, the protein looses its haemostatic function, and thus its therapeutic effect. The degradation rate is proportional to the concentration of the activated Factor VII polypeptide in the second order as follows:−d[FVIIa]/dt=k[FVIIa]2 where [FVIIa] is the concentration of the activated Factor VII polypeptide.
This means that if the activated Factor VII polypeptide is concentrated e.g. 10 times, then the degradation rate is expected to increase 100 times.
Another difficulty of concentrating an activated Factor VII polypeptide is its solubility, which means that the activated Factor VII polypeptide will start to precipitate when it reaches a certain concentration in any given buffer.